Inhibins are members of the transforming growth factor β (TGF-β) superfamily and are dimeric in structure (Evans and Groome (2001) Development of Immunoassays for Inhibin, Activin and Follistatin. In: S. Muttukrishna and W. Ledger (Eds.) Inhibin, Activin and Follistatin in Human Reproductive Physiology. [Imperial College Press, London], pp. 11-60; Robertson et al. (2004) Endocr. Relat. Cancer 11:35; Robertson et al. (2004) Mol. Cell. Endocrinol. 225:65). They regulate the reproductive system by acting on the pituitary gland and blocking the synthesis of the FSH-β subunit and therefore the secretion of FSH (Burger and Igarashi (1988) J. Clin. Endocrinol. Metab. 66:885; Attardi et al. (1992) Endocrinol. 130:557; Burger et al. (1998) J. Clin. Endocrinol. Metab. 83:4167; Knight and Glister (2001) Reproduction 121:503). Inhibins are heterodimeric molecules containing an β subunit and either a βA or βB subunit, which are connected to each other by a disulfide bond. If the dimer consists of a βA subunit the molecule is called inhibin A, and if it consists of a βB subunit the molecule is called inhibin B (Miyamoto et al. (1985) Biochem. Biophys. Res. Commun. 129:396; Robertson et al. (1985) Biochem. Biophys. Res. Commun. 126:220; Mason et al. (1986) Biochem. Biophys. Res. Commun. 135:957). Activins contain two β subunits and can be homodimeric or heterodimeric depending on the arrangement of their subunits (Ling et al. (1986) Biochem. Biophys. Res. Commun. 138:1129; Vale et al. (1986) Nature 321:776). Two βA subunits make activin A, two βB subunits make activin B and a βA subunit attached to a βB subunit make activin AB (Vale et al., 1986; Vale et al. (1988) Recent Prog. Horm. Res. 44:1).
The measurement of inhibins in biological fluids has led to insights into its physiology, such as the pattern of inhibins in the menstrual cycle (Groome et al. (1996) J. Clin. Endocrinol. Metab. 81:1401). Some of the many applications include; Down's syndrome screening (inhibin A), male infertility testing (inhibin B), ovarian reserve/menopause onset (inhibin B) and ovarian cancer (inhibin αC subunit and inhibin B) (Illingworth et al. (1996) J. Clin. Endocrinol. Metab. 81:1321; Muttukrishna et al. (2000) Hum. Reprod. 15:549; Robertson et al. (2002) J. Clin. Endocrinol. Metab. 87:816; Wald et al. (2003) Lancet 361:835).
Two commercial inhibin B immunoassays are available, from DSL and OBI. Both assays use the same pair of monoclonal antibodies raised to synthetic peptides generated over 10 years ago. The capture antibody (C5), was raised to a peptide from the βB subunit of inhibin, and the detection antibody (R1) was raised to a peptide from the α subunit of inhibin. Both assays require a methionine oxidation step with hydrogen peroxide to allow the C5 antibody to recognize its epitope, which contains the amino acid sequence MSM. The original use of an oxidation step was used by Knight and Muttukrishna (1994) J. Endocrinol. 141:417, who showed that it improved sensitivity of the inhibin A assay, and subsequently improved the activin A assay (Knight et al. (1996) J. Endocrinol. 148:267). Hydrogen peroxide is used to oxidize methionine to methionine sulfoxide in order for C5 to bind. The OBI assay also uses a SDS and heat pre-treatment (Wallace et al. (1998) Ann. Clin. Biochem. 35:656) of the sample to destroy any catalases that may affect the oxidation step (especially in haemolysed samples), destroy any proteases that may be present, remove binding proteins and remove potential false positive causing reagents (Evans and Groome, 2001). The OBI and DSL assays have a detection limit of approximately 7 pg/ml, and the cross-reactivity of the inhibin B assays with inhibin A was initially reported as approximately 0.5% (Groome et al., 1996). Adequate sensitivity of the present assays requires overnight incubation with the sample.
Several unsuccessful attempts to generate a superior replacement for the C5 antibody using synthetic peptide immunogens prompted the present alternative approach. The present invention is based on part on the discovery and development of antibodies to the βB subunit of inhibin/activin which are superior tools for immunoassay and immunohistochemistry.